The attachment of dissimilar materials is a major challenge because of the high levels of localized stress that develop at such interfaces. An effective biologic solution to this problem develops post-natally at the attachment of tendon to bone. This tissue, the tendon enthesis, transitions from tendon to fibrocartilage to mineralized fibrocartilage to bone across a short distance. The enthesis is not recreated during tendon-to-bone healing. Surgical reattachment of these two dissimilar biologic materials therefore often fails. A better understanding of tendon enthesis development will allow us to recapitulate development for tendon-to-bone repair and enthesis tissue engineering. A number of studies have shown a role for mechanobiology in fetal joint development and in adult joint homeostasis. However, few studies have examined the role of mechanical loading on joint development in the early post-natal period. The mechanobiology of post-natal development is especially relevant with respect to the shoulder. Neonatal brachial plexus palsy due to injuries at childbirth can lead to a number of shoulder pathologies in children. These conditions are thought to be a result of abnormal rotator cuff muscle loading across the developing joint. Little is known about the mechanobiology that drives post-natal shoulder development, so clinical options for these patients is limited. We propose to study the mechanobiology of tendon enthesis development using mouse models. We recently published a mouse model in which rotator cuff muscles were paralyzed at birth using an injection of botulinum toxin and paralysis was maintained until sacrifice. We demonstrated that muscle loading is necessary for post-natal tendon enthesis development. In this proposal, we will use normal and genetically modified mouse models to examine the mechanical and molecular factors that influence the development of the unique tissue at the attachment of tendon to bone. Our first set of aims will examine the role of three molecules thought to play important roles for differentiation and mechanotransduction of osteoblasts, fibrochondrocytes, and tendon fibroblasts (i.e., the three main cell types found at the enthesis). We will examine the role of connexin43, Indian hedgehog, and scleraxis for bone, fibrocartilage, and tendon formation, respectively, at the enthesis. Our second set of aims will examine the potential for rescuing the defects caused by muscle paralysis during post-natal development. We will attempt to rescue the bony defects by suppressing osteoclasts activity with bisphosphonates and promoting osteoblast activity with PTH. We will also study the recovery potential of the developing tendon enthesis subjected to varying durations of paralysis and recovery.